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Results for “isopeptide bonds”

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At least 19 recordsLinked to original sources
PubMed2025

Type IV Pili-Associated Secretion of a Biofilm Matrix Protein From Clostridium perfringens That Forms Intermolecular Isopeptide Bonds.

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterial pathogen of humans and animals. C. perfringens also produces type IV pili (T4P) and has two complete sets of T4P-associated genes, one of which has been shown to produce surface pili needed for cell adherence. One hypothesis about the second set of T4P genes is that they comprise a type II secretion system (TTSS) like those found in gram-negative bacteria, but for gram-positive bacteria, the TTSS would aid transit across the thick peptidoglycan (PG) layer. The secretome of mutants lacking type IV pilins was examined, and a single protein, BsaC (CPE0517), was identified as being dependent on pilin PilA3 for secretion. The bsaC gene is in an operon with genes encoding a SipW signal peptidase and two putative biofilm matrix proteins, BsaA and BsaB, both of which have remote homology to Bacillus subtilis biofilm protein TasA. Since BsaA forms long oligomers that are secreted, we analyzed BsaA monomer interactions with de novo modeling. These models projected that the monomers formed isopeptide bonds as part of a donor strand exchange process. Mutations in residues predicted to form the isopeptide bonds led to the loss of oligomerization, supporting an exchange and lock mechanism, and isopeptide bonds were detected by mass spectrometry methods. Phylogenetic analysis showed the BsaA family of proteins is widespread among bacteria and archaea, but only a subset is predicted to form isopeptide bonds.

Fimbriae, Bacterial↗
PubMed1976

[On the occurence of isopeptide bonds in the chorion of the rainbow trout (Salmo gairdneri Rich.) (author's transl)].

Chorions of unfertilized and fertilized eggs of the rainbow trout were isolated from tissue and yolk components and investigated for isopeptide bonds. The complete hydrolysis of the alpha-amide bonds was obtained by a system of four proteases. A chromatographic ion exchange system was used to separate Nepsilon-(beta-aspartyl)lysine as well as Nepsilon-(gamma-glutamyl)lysine from single amino acids. Chorions of unfertilized eggs contain neither Asp Lys nor Glu Lys bonds. Only chorions of fertilized eggs contain Glu Lys isopeptide. It is probably the high content of this isopeptide that is responsible for the greater mechanical stability of the fertilized teleostean egg chorion.

Amino Acids↗
PubMed1976

[On the occurence of isopeptide bonds in heated milk protein (author's transl)].

Milk, milk products and individual milk proteins were subjected to different heat treatments either as powders or in aquous systems. After complete hydrolysis of the peptide bonds (alpha-amide bonds) by a system of four proteinases or peptidases, respectively, the samples were analysed for isopeptides. For this purpose, two chromatographic ion exchange systems were developed, each of which separates Nepsilon-(beta-aspartyl-)lysine (Asp Lys) as well as Nepsilon-(gamma-glutamyl-)lysine (Glu Lys) from the common amino acids. In samples, heated 24 h for at least 120 degrees C, 2-5% of the lysine residues are incorporated in Glu Lys-bonds. Under the heating conditions used in dairy practice, no isopeptide bonds were formed.

Animals↗
PubMed1977

The enzymic derivation of citrulline residues from arginine residues in situ during the biosynthesis of hair proteins that are cross-linked by isopeptide bonds.

An enzymic activity present in hair follicles is described that can convert arginine residues to citrulline residues in proteins in situ. The Ca2+ dependent enzyme activity has been detected in hair follicle extracts but not in similar extracts of serum, liver or brain. The enzyme appears to act on proteins other than hair proteins and the citrulline produced can be quantitated in acid hydrolysates by a colorimetric procedure. The formation of citrulline has been confirmed by amino acid analysis and does not appear to be related to the formation of isopeptide linkages which is catalysed by the transamidase present in hair follicles.

Amino Acids↗
PubMed1978

A method for improving the nutritional value of food proteins: covalent attachment of amino acids.

Casein was modified by use of a series of active N-hydroxy-succinimide esters of amino acids in order to study the effects of new covalently linked hydrophobic or hydrophilic groups on its physical and nutritional properties. Tryptophan was used to determine the best conditions for the chemical reaction and to study the stability of the newly formed amide linkage (isopeptide bond). Casein was also modified with glycine, alanine, methionine, N-acetyl-methionine and aspartic acid. In vitro hydrolysis studies using bovine chymotrypsin, pancreatine and rat bile-pancreatic juice indicated that digestibility of the modified casein derivatives was lower than that of the untreated protein. Since solubility was not significantly changed (except for tryptophyl-casein), the decreased in vitro digestibility is probably due to other factors such as steric hindrance as well as decrease in lysine residues available to trypsin in pancreatin and rat pancreatic juice. Plasma amino acid patterns for rats fed a 10% protein diet of highly modified glycyl-casein or methionyl-casein suggest that the epsilon-aminolysyl derivatives are readily hydrolyzed in vivo. This was confirmed by the growth response of rats fed the following isonitrogenous diets (protein source listed only): casein, casein + free methionine, methionyl-casein, casein + free N-acetyl-methionine, N-acety-methionyl-casein. Covalently attached methionine appeared to be as readily available as the free amino acid; bound N-acetyl-methionine was also available but to a slightly lower extent. Although this study is preliminary, the covalent attachment of amino acids to proteins appears to be a promising method for improving the biological value of food proteins.

Amino Acids↗
PubMed1977

Heat induced crosslinks in milk proteins and consequences for the milk system.

The protein system of milk is rather unusual, there are nearly no interchain crosslinks found. Even intrachain crosslinks, especially disulfide bridges, are present only in about every fourth protein molecule. Heating causes dramatic changes in the structure of milk proteins, resulting in the formation of polymeric networks. The contribution of individual milk proteins, namely the beta-lactoglobulins, alpha-lactalbumin and chi-casein, to the formation of crosslinks is studied with respect to heating temperature and time, pH and atmosphere. Measured are changes in molecular weights and in the SH/SS-levels as well as the formation of dehydroalanine, lysinoalanine, lanthionine and isopeptide bonds. Some practical aspects of crosslinking in milk proteins are discussed.

Animals↗
PubMed1977

Fibrin digestion by thrombin. Comparison with plasmin-digested fibrinogen.

Solutions of plasminogen-free human fibrinogen alone or (1) treated with sodium p-chloromercuribenzoate in order to inactivate factor XIII, or (2) enriched with factor XIII, cysteine and CaC12, were clotted with plasmin-free human thrombin and incubated under sterile conditions. The clots dissolved gradually within 2 days (fibrin from sodium p-chloromercuribenzoate-treated fibrinogen) to 15 days (fibrin from factor XIII-enriched fibrinogen). This proteolytic process was not affected by soybean trypsin inhibitor but was completely inhibited by hirudin. Gel electrophoresis of the thrombin digests indicated the formation of bands equivalent to bands X, Y, D and E of plasmin digests of fibrinogen. The two latter bands, whose identity was confirmed by immunoelectrophoresis, appeared at a more advanced stage of proteolysis than the corresponding bands of plasmin digests. The number of isopeptide bonds present did not appear to affect the rate of release of acid-soluble peptides. Gel electrophoresis and the rate of release of acid-soluble peptides indicated that fewer bonds are hydrolysed by thrombin at the time of the complete solubilization of the clot than are split by plasmin when fibrinogen becomes unclottable by thrombin.

Blood Coagulation↗
PubMed1992

Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development.

Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.

Animals↗
PubMed1992

Molecular conformation of ubiquitinated structures and the implications for regulatory function.

The molecular conformation of ubiquitinated structures and the validity of the N-end rule were examined by simulating the molecular mechanics to ascertain the global energy-minimized structure. We examined the chemical linkage involved in attaching the ubiquitin carboxyl terminus to the N-terminus of three different x-hexapeptides, where x is the amino group of the acceptor peptide--either valine, arginine or glutamic acid--(x-K linkage) and to the epsilon-amino group of lysine of the acceptor hexapeptide x-glu1-his2-lys3-gly4-lys5-val6 (K-K linkage) through the formation of an isopeptide bond. Changes in conformation and molecular stability of the multi-ubiquitinated structures were determined by energy-minimization procedures using the SYBYL program developed by Tripos Associates. In the x-K linkage, the ubiquitin molecule is stretched in the beta-pleated sheets and beta-turns while the alpha-helices expand, as the molecule continues to unfold linearly. In the K-K linkage, the ubiquitin molecules have turned into a u-shaped, semi-circular alignment, contracting into a compact, folded structure.

Amino Acid Sequence↗
PubMed1977

Isopeptide linkage between N-alpha-monomethylalanine and lysine in ribosomal protein S11 from Escherichia coli.

Protein S11 from the Escherichia coli ribosome has a unique NH2-terminal structure not previously observed among ribosomal proteins. Owing to the formation of an isopeptide bond between a secondary amino acid (N-alpha-monomethylalanine) and the epsilon-amino group of the NH2-terminal lysine residue, a "branching point" is formed. Therefore, two amino acids are seen when the NH2 terminus of the protein is determined.

Alanine↗
PubMed1977

Isopeptide linkage between nonhistone and histone 2A polypeptides of chromosomal conjugate-protein A24.

Chromosomal protein A24 has a unique structure inasmuch as it contains histone 2A and a nonhistone polypeptide the sequence of which has been partially determined. Comparative analysis of the ninhydrin-insensitive amino-terminal tryptic peptides of protein A24 and histone 2A and a quantitative analysis of their carboxyl-terminal amino acid indicated that protein A24 has two amino termini and one carboxyl terminus. The amino acid sequence analysis of tryptic peptide 17' of protein A24: (see text) showed it contains tryptic peptide 17 of histone 2A, Lys-Thr-Glu-Ser-His-His-Lys. Lysine 119, the amino terminus of this peptide, which is derived from the histone 2A portion of protein A24, is linked by an isopeptide bond to the carboxyl group of a glycine residue. Accordingly, the branched structure of protein A24 proposed is: (see text).

Amino Acid Sequence↗
PubMed2026

Analytical challenges for mapping non-canonical and non-protein ubiquitin/Ubl modifications by mass spectrometry.

INTRODUCTION: Covalent modification by ubiquitin via Lys isopeptide bonds is fundamental for regulating protein turnover and function. Additionally, ubiquitin esterification occurs on Ser/Thr/Tyr residues in proteins and on non-proteinaceous substrates including ribose, saccharides, lipids, and small molecule drugs. Ubiquitin posttranslational modifications may therefore be much more widespread across cell biological pathways. Recent literature (PubMed) reflects the increased interest in analytical methods for mapping of non-canonical substrates modified by ubiquitin and ubiquitin-like (UBL) proteins. AREAS COVERED: Mass spectrometry (MS)-based methodologies involve advanced proteomic techniques to identify ubiquitin modifications on amino acids other than Lys, such as Ser, Thr, Tyr and Cys as well as protein N-termini. After digestion, standard MS workflows identify canonical ubiquitination by detecting a ubiquitin C-terminal tag attached to the amine side chains of Lys residues of substrate-derived peptides suitable for MS/MS sequencing. For non-canonical modifications on proteins and substrates other than proteins, specialized strategies are required, such as using antibodies to enrich N-terminally modified peptides in combination with using high-resolution MS/MS based on softer fragmentation technologies to detect esterification and possibly other types of substrate modifications. EXPERT OPINION: Enabling such technologies will reveal a previously unrecognized angle of the ubiquitin code's complexity in cells.

Humans↗
PubMed1992

Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2).

Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. In such multiubiquitin chains, successive ubiquitins are linked by an isopeptide bond involving the side chain of Lys48 and the carboxyl group of Gly76. The crystal structure of a diubiquitin conjugate has been determined and refined at 2.3-A resolution. The molecule has internal approximate 2-fold symmetry with multiple hydrophobic and hydrophilic contacts along the 2-fold axis. The structure of the diubiquitin conjugate suggests determinants for recognition of multiubiquitin chains. A model for the interaction of diubiquitin and a ubiquitin conjugating enzyme (E2) is proposed.

Amino Acid Sequence↗
PubMed1992

Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K).

Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next. During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination). Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59. The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin. The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59. Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons. Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase. These findings should assist in the design of future studies concerning E2(25K) structure and function.

Amino Acid Sequence↗
PubMed1978

[Utilization of pure proteins and peptides for the study of chemical and nutritional changes suffered by milk during heating].

Milk proteins, and in particular the caseins, undergo during heat treatments (120 degrees C, 20-30 min) physico chemical and nutritional modifications. Using pure proteins (alphas and beta caseins) and peptides, it has been possible to dissociate the effects of heat treatments which, in milk can hide each other. Physico chemical properties of caseins (the ability to bind Ca++ and anionic dyes, acid-basic titration curves, electrophoretic behavior) are strongly altered, while the constitutive aminoacids are less modified. The accessibility of the aminogroups of lysine to the fluorodinitrobenzen and to the O-méthylisourea is lowered with a small rate. Lysinoalanyle interactions between serine and lysine are weak at a neutral pH while the isopeptide bonds epsilonN (gamma glutamyl)lysyle are only produced with more severe conditions as those of sterilization. The digestibility to the proteases is enhanced with low heat treatments then decreases with more severe treatments (120 degrees C-80 min.). Many peptides, which are released from the caseins during heating are issued from low specific split peptidic bonds; these peptides have often stimulating properties on the growth of lactic bacteria.

Amino Acids↗
PubMed1979

Covalent bonds formed in proteins during milk sterilization: studies on caseins and casein peptides.

Formation of covalent bonds at milk sterilization temperatures was studied using caseins and casein peptides. At 120 degrees C lysinoalanyl residues produced even at pH 7.0 were derived from intra-molecular interactions between phosphoserine and lysine; the conditions of formation were determined. It was also found that the formation fo isopeptidic cross-links was significant with conditions more severe than those used for milk sterilization.

Amino Acids↗
PubMed1976

Comparison of the physicochemical properties of fragment D derivatives of fibrinogen and fragment D-D of cross-linked fibrin.

The molecular weight of Fragment D derivatives obtained from plasmic digests of fibrinogen and cross-linked fibrin was determined by equilibrium sedimentation and compared with the summated molecular weight of their polypeptide chains observed after electrophoresis of reduced protein in sodium dodecyl sulfate polyacrylamide gels. The measured molecular weight of Fragment D (Stage 2) of fibrinogen is 103 500, which is compatible with a molecule containing only one each of the Aalpha (13 000), Bbeta (43 000) and gamma (39 000) chain remnants. Fragment D-D of cross-linked fibrin has a molecular weight of 189 000, compatible with a molecule containing one isopeptide-bound gamma-gamma chain (80 000) and two each of Bbeta (43 000) and Aalpha (13 000) chain remnants. The NH2-terminal amino acid residues of the Fragment D derivatives were measured quantitatively using a thioacetic-thioglycolic acid method, and molar quantities were calculated on the basis of the molecular weights determined by equilibrium sedimentation. Fragment D preparations obtained from Stage 2 and Stage 3 digests of fibrinogen have 3 mol of NH2-terminal amino acids per molecule, while Fragment D-D has seven. These data support the view that two Fragment D molecules, each of three polypeptide chains, are derived by plasmic degradation from each fibrinogen molecule, and that an isopeptide-bound, six chain Fragment D-D molecule is released from cross-linked fibrin by plasmin. Equlibrium sedimentation measurement of the molecular weights of Fragment X (Stage 1 and Stage 2) and Fragment Y are 265 000 and 148 000, respectively. These values are compatible with asymmetric cleavages of Fragment X to Fragments Y and D (Stage 2), and of Fragment Y to Fragments D (Stage 2) and E, and with a fibrinogen model in which the two halves are joined by disulfide bonds only in the amino-terminal regions.

Electrophoresis, Polyacrylamide Gel↗